This CRCNS grant application proposes structure, function, and dynamic studies on the plasma membrane dopamine (DAT) and serotonin transporters (SERT). Recently, the arduous process of identifying DAT and SERT substrate and inhibitor binding sites received an unexpected boost with the crystallization of the homologous LeuTAa leucine transporter. This crystal structure revealed hinged regions of transmembranes (TMs) 1 and 6 adjacent to the leucine substrate, with TMs 3, 8 and 10 also delineating the binding pocket. Through comparative molecular modeling techniques we have published a three-dimensional model of DAT using LeuTAa. The modeling also suggests novel inhibitor binding sites, nonidentical to dopamine, that can be tested via molecular pharmacological techniques. Intellectual Merit: This project brings together a unique team of computational scientists, medicinal chemists, and pharmacologists to examine the structure, function, and dynamics of monoamine neurotransporters (MATs). The overall goal of this project is to determine binding locations for psychostimulant and antidepressant inhibitors of neurotransmitter transport, and the conformational states involved in the transport mechanism. State-of-the-art computational techniques in areas of docking, advanced molecular dynamics simulations, QSAR and structure-based design will be used to identify important residues and regions of the transporter to perform mutagenesis experiments as well as the direct the synthesis of novel compounds that inhibit binding of non-neurotransmitter molecules yet retain transporter activity. Broader Impacts: The impacts of this proposal fall into three categories: (1) the scientific community, (2) the institution, and (3) the individual research groups. The scientific community will benefit from the results of this project through the availability of validated and tested three-dimensional structures of DAT and SERT. These structures can then be used in structure-based design, which is an advance on current ligand-based design efforts. The resultant 3-D transporter "blueprint" can be used to screen databases for novel MAT ligands. How specific MAT protein components contribute to discrimination between, for example, cocaine (high abuse potential) and methylphenidate (low abuse potential) may be elucidated. This is a critical health issue, as the search for therapeutically useful compounds in combating psychostimulant abuse and addiction has been in progress for decades, with no clinically available agents to date. The proposed work may open the door to rational design of therapeutics for MAT-related conditions that include substance abuse and addiction, depression, anxiety disorders, attention deficit hyperactivity disorder, narcolepsy, chronic pain and Parkinson's disease. The community will also benefit from the addition of newly graduated scientists cross-trained in computational and experimental neurosciences. The impact at the institution will be increased research infrastructure and promotion of multi-disciplinary research activities. The impact of this proposal on the individual research teams will be to enhance their efforts to select from a talented and diverse pool of students. Currently the combined groups have five male and four female students. These students come from Argentina, China, India, and the United States. We will also continue to offer a molecular modeling course in which several of the current students of the research team have already taken. New material for the molecular modeling course will be drawn from our experiences and results from this project. Finally the PI is a general chemistry textbook author and results from this work will be incorporated into the textbook. In summary, this proposal fits the criteria for this program in the following manner: (1) it includes collaborations between computational and/or modeling experts (Madura research group), experimental neuropharmacologists (Surratt research group) and medicinal chemists (Lapinsky research group);(2) this collaboration involves intense, dynamic interactions among these research groups in the model development and refinement of dopamine and serotonin neurotransporters;and (3) leading to the development and testing of new models that provide a framework for the design of experiments and the generation of new hypotheses to reveal mechanisms underlying normal or diseased states of the nervous system.